Fig. 2

Immunoblot and RT-PCR analysis of BIN1 isoform diversity in the human brain. a The schematic structure of BIN1 indicating the epitopes of the antibodies used in this study. b Immunoblot analysis of neuronal and oligodendrocyte marker protein levels in extracts prepared from the human brain gray matter and white matter. c Immunoblot analysis of BIN1 and Amphiphysin 1 levels. The pAb BSH3 reacts with all BIN1 isoforms whereas mAb 2 F11 and 99D react with D7-BIN1 and + exon 17 BIN1 isoforms, respectively. The asterisks indicate non-specific signals. d The signal intensities of BIN1 were quantified using the Li-COR system, normalized to actin and plotted as mean ± SEM (n = 8). Statistical significance was assessed by ANOVA. e Schematic illustration of the alternatively spliced human brain BIN1 isoforms identified in this study. Five independent human brain samples were subjected to RT-PCR analysis by using primers corresponding to sequences within exons 12 and 18 and the resulting amplicons were fractionated by electrophoresis. The bands were excised and individually sequenced to identify the major isoforms generated by alternate splicing of exons 13–17. The exclusion of BIN1 exon 11 in brain and inclusion of exon 7 in isoforms 1 and 3 were inferred from the published literature and the RT-PCR and immunoblot results of our study